Alterations in Cholesterol and Phosphoinositides Levels in the Intracellular Cholesterol Trafficking Disorder NPC.

Lipid mistrafficking is a biochemical hallmark of Niemann-Pick Type C (NPC) disease and is classically characterized with endo/lysosomal accumulation of unesterified cholesterol due to genetic mutations in the cholesterol transporter proteins NPC1 and NPC2. Storage of this essential signaling lipid leads to a sequence of downstream events, including oxidative stress, calcium imbalance, neuroinflammation, and progressive neurodegeneration, another hallmark of NPC disease. These observations have been validated in a growing number of studies ranging from NPC cell cultures and animal models to patient specimens. In recent reports, alterations in the levels of another class of critical signaling lipids, namely phosphoinositides, have been described in NPC disease. Focusing on cholesterol and phosphoinositides, the chapter begins by reviewing the interactions of NPC proteins with cholesterol and their role in cholesterol transport. It then continues to describe the modulation of cholesterol efflux in NPC disease. The chapter concludes with a summary of findings related to the functional consequences of perturbations in phosphoinositides in this fatal disease.

Waning efficacy in a long-term AAV-mediated gene therapy study in the murine model of Krabbe disease.

Neonatal AAV9-gene therapy of the lysosomal enzyme galactosylceramidase (GALC) significantly ameliorates central and peripheral neuropathology, prolongs survival, and largely normalizes motor deficits in Twitcher mice. Despite these therapeutic milestones, new observations identified the presence of multiple small focal demyelinating areas in the brain after 6-8 months. These lesions are in stark contrast to the diffuse, global demyelination that affects the brain of naive Twitcher mice. Late-onset lesions exhibited lysosomal alterations with reduced expression of GALC and increased psychosine levels. Furthermore, we found that lesions were closely associated with the extravasation of plasma fibrinogen and activation of the fibrinogen-BMP-SMAD-GFAP gliotic response. Extravasation of fibrinogen correlated with tight junction disruptions of the vasculature within the lesioned areas. The lesions were surrounded by normal appearing white matter. Our study shows that the dysregulation of therapeutic GALC was likely driven by the exhaustion of therapeutic AAV episomal DNA within the lesions, paralleling the presence of proliferating oligodendrocyte progenitors and glia. We believe that this is the first demonstration of diminishing expression in vivo from an AAV gene therapy vector with detrimental effects in the brain of a lysosomal storage disease animal model. The development of this phenotype linking localized loss of GALC activity with relapsing neuropathology in the adult brain of neonatally AAV-gene therapy-treated Twitcher mice identifies and alerts to possible late-onset reductions of AAV efficacy, with implications to other genetic leukodystrophies.

Mass spectrometry-based lipid analysis and imaging.

Mass spectrometry imaging (MSI) is a powerful tool for in situ mapping of analytes across a sample. With growing interest in lipid biochemistry, the ability to perform such mapping without antibodies has opened many opportunities for MSI and lipid analysis. Herein, we discuss the basics of MSI with particular emphasis on MALDI mass spectrometry and lipid analysis. A discussion of critical advancements as well as protocol details are provided to the reader. In addition, strategies for improving the detection of lipids, as well as applications in biomedical research, are presented.

Mass spectrometry imaging and LC/MS reveal decreased cerebellar phosphoinositides in Niemann-Pick type C1-null mice.

Niemann-Pick disease type C1 (NPC1) is a lipid storage disorder in which cholesterol and glycosphingolipids accumulate in late endosomal/lysosomal compartments because of mutations in the NPC1 gene. A hallmark of NPC1 is progressive neurodegeneration of the cerebellum as well as visceral organ damage; however, the mechanisms driving this disease pathology are not fully understood. Phosphoinositides are phospholipids that play distinct roles in signal transduction and vesicle trafficking. Here, we utilized a consensus spectra analysis of MS imaging data sets and orthogonal LC/MS analyses to evaluate the spatial distribution of phosphoinositides and quantify them in cerebellar tissue from Npc1-null mice. Our results suggest significant depletion of multiple phosphoinositide species, including PI, PIP, and PIP2, in the cerebellum of the Npc1-null mice in both whole-tissue lysates and myelin-enriched fractions. Additionally, we observed altered levels of the regulatory enzyme phosphatidylinositol 4-kinase type 2α in Npc1-null mice. In contrast, the levels of related kinases, phosphatases, and transfer proteins were unaltered in the Npc1-null mouse model, as observed by Western blot analysis. Our discovery of phosphoinositide lipid biomarkers for NPC1 opens new perspectives on the pathophysiology underlying this fatal neurodegenerative disease.

Mass spectrometry imaging reveals ganglioside and ceramide localization patterns during cerebellar degeneration in the Npc1-/- mouse model.

Mass spectrometry imaging (MSI) is a powerful tool to perform untargeted mapping of biomolecules in situ. In the current study, we performed matrix-assisted laser desorption/ionization-mass spectrometry imaging (MALDI-MSI) to evaluate lipid changes during disease progression (asymptomatic to symptomatic time points) in Niemann-Pick disease, type C1 (NPC1), a cerebellar neurodegenerative, lipid storage disorder. Our data show that gangliosides GM2 and GM3 are elevated in NPC1 disease and localize in the posterior lobules of the cerebellum, which is enhanced over a time-course analysis of the disease. Further analysis of sphingolipids in negative ion mode indicated reduction of sulfatides in white matter of the cerebellum and patterned distribution and co-localization of ceramide species Cer(d36:1), HexCer(d36:1), and the ganglioside GM1(d36:1) during disease progression. Finally, a putative lipid of unknown structure demonstrated similar patterning during NPC1 cerebellar degeneration. These studies provide insight into lipid markers of neurodegeneration in NPC1 and link lipid alterations to altered pathways that lead to cell death.

Gel-assisted mass spectrometry imaging enables sub-micrometer spatial lipidomics.

A technique capable of label-free detection, mass spectrometry imaging (MSI) is a powerful tool for spatial investigation of native biomolecules in intact specimens. However, MSI has often been precluded from single-cell applications due to the spatial resolution limit set forth by the physical and instrumental constraints of the method. By taking advantage of the reversible interaction between the analytes and a superabsorbent hydrogel, we have developed a sample preparation and imaging workflow named Gel-Assisted Mass Spectrometry Imaging (GAMSI) to overcome the spatial resolution limits of modern mass spectrometers. With GAMSI, we show that the spatial resolution of MALDI-MSI can be enhanced ~3-6-fold to the sub-micrometer level without changing the existing mass spectrometry hardware or analysis pipeline. This approach will vastly enhance the accessibility of MSI-based spatial analysis at the cellular scale.

Mutant induced neurons and humanized mice enable identification of Niemann-Pick type C1 proteostatic therapies.

Therapeutics that rescue folding, trafficking, and function of disease-causing missense mutants are sought for a host of human diseases, but efforts to leverage model systems to test emerging strategies have met with limited success. Such is the case for Niemann-Pick type C1 disease, a lysosomal disorder characterized by impaired intracellular cholesterol trafficking, progressive neurodegeneration, and early death. NPC1, a multipass transmembrane glycoprotein, is synthesized in the endoplasmic reticulum and traffics to late endosomes/lysosomes, but this process is often disrupted in disease. We sought to identify small molecules that promote folding and enable lysosomal localization and functional recovery of mutant NPC1. We leveraged a panel of isogenic human induced neurons expressing distinct NPC1 missense mutations. We used this panel to rescreen compounds that were reported previously to correct NPC1 folding and trafficking. We established mo56-hydroxycholesterol (mo56Hc) as a potent pharmacological chaperone for several NPC1 mutants. Furthermore, we generated mice expressing human I1061T NPC1, a common mutation in patients. We demonstrated that this model exhibited disease phenotypes and recapitulated the protein trafficking defects, lipid storage, and response to mo56Hc exhibited by human cells expressing I1061T NPC1. These tools established a paradigm for testing and validation of proteostatic therapeutics as an important step toward the development of disease-modifying therapies.

DBDA matrix increases ion abundance of fatty acids and sulfatides in MALDI-TOF and mass spectrometry imaging studies.

MALDI-TOF MS is a powerful tool to analyze biomolecules owing to its soft ionization nature and generally results in simple spectra of singly charged ions. Moreover, implementation of the technology in imaging mode provides a means to spatially map analytes in situ. Recently, a new matrix, DBDA (N1,N4-dibenzylidenebenzene-1,4-diamine) was reported to facilitate the ionization of free fatty acids in the negative ion mode. Building on this finding, we sought to implement DBDA for MALDI mass spectrometry imaging studies in brain tissue and successfully map oleic acid, palmitic acid, stearic acid, docosahexaenoic acid and arachidonic acid using mouse brain sections. Moreover, we hypothesized that DBDA would provide superior ionization for sulfatides, a class of sulfolipids, with multiple biological functions. Herein we also demonstrate that DBDA is ideal for MALDI mass spectrometry imaging of fatty acids and sulfatides in brain tissue sections. Additionally, we show enhanced ionization of sulfatides using DBDA compared to three different traditionally used MALDI matrices. Together these results provide new opportunities for studies to measure sulfatides by MALDI-TOF MS including in imaging modes.

Gel-assisted mass spectrometry imaging.

Compatible with label-free detection and quantification, mass spectrometry imaging (MSI) is a powerful tool for spatial investigation of biomolecules in intact specimens. Yet, the spatial resolution of MSI is limited by the method's physical and instrumental constraints, which often preclude it from single-cell and subcellular applications. By taking advantage of the reversible interaction of analytes with superabsorbent hydrogels, we developed a sample preparation and imaging workflow named Gel-Assisted Mass Spectrometry Imaging (GAMSI) to overcome these limits. With GAMSI, the spatial resolution of lipid and protein MALDI-MSI can be enhanced severalfold without changing the existing mass spectrometry hardware and analysis pipeline. This approach will further enhance the accessibility to (sub)cellular-scale MALDI-MSI-based spatial omics.

DBDA Matrix Increases Ion Abundance of Fatty Acids and Sulfatides in MALDI-TOF and Mass Spectrometry Imaging Studies.

MALDI-TOF MS is a powerful tool to analyze biomolecules, owing to its soft ionization nature that generally results in simple spectra of singly charged ions. Implementation of the technology in the imaging mode provides a means to spatially map analytes in situ. Recently, a new matrix, DBDA (N1,N4-dibenzylidenebenzene-1,4-diamine) was reported to facilitate the ionization of free fatty acids in negative ion mode. Building on this finding, we sought to implement DBDA for MALDI mass spectrometry imaging studies in brain tissue and successfully map oleic acid, palmitic acid, stearic acid, docosahexaenoic acid, and arachidonic acid using mouse brain sections. Moreover, we hypothesized that DBDA would provide superior ionization for sulfatides, a class of sulfolipids with multiple biological functions. Herein, we also demonstrate that DBDA is ideal for MALDI mass spectrometry imaging of fatty acids and sulfatides in brain tissue sections. Additionally, we show enhanced ionization of sulfatides using DBDA compared with three different traditionally used MALDI matrices. Together these results provide new opportunities for studies to measure sulfatides by MALDI-TOF MS.

CD36 maintains lipid homeostasis via selective uptake of monounsaturated fatty acids during matrix detachment and tumor progression.

A high-fat diet (HFD) promotes metastasis through increased uptake of saturated fatty acids (SFAs). The fatty acid transporter CD36 has been implicated in this process, but a detailed understanding of CD36 function is lacking. During matrix detachment, endoplasmic reticulum (ER) stress reduces SCD1 protein, resulting in increased lipid saturation. Subsequently, CD36 is induced in a p38- and AMPK-dependent manner to promote preferential uptake of monounsaturated fatty acids (MUFAs), thereby maintaining a balance between SFAs and MUFAs. In attached cells, CD36 palmitoylation is required for MUFA uptake and protection from palmitate-induced lipotoxicity. In breast cancer mouse models, CD36-deficiency induced ER stress while diminishing the pro-metastatic effect of HFD, and only a palmitoylation-proficient CD36 rescued this effect. Finally, AMPK-deficient tumors have reduced CD36 expression and are metastatically impaired, but ectopic CD36 expression restores their metastatic potential. Our results suggest that, rather than facilitating HFD-driven tumorigenesis, CD36 plays a supportive role by preventing SFA-induced lipotoxicity.

Species-specific differences in NPC1 protein trafficking govern therapeutic response in Niemann-Pick type C disease.

The folding and trafficking of transmembrane glycoproteins are essential for cellular homeostasis and are compromised in many diseases. In Niemann-Pick type C disease, a lysosomal disorder characterized by impaired intracellular cholesterol trafficking, the transmembrane glycoprotein NPC1 misfolds due to disease-causing missense mutations. While mutant NPC1 has emerged as a robust target for proteostasis modulators, drug development efforts have been unsuccessful in mouse models. Here, we demonstrated unexpected differences in trafficking through the medial Golgi between mouse and human I1061T-NPC1, a common disease-causing mutant. We established that these distinctions are governed by differences in the NPC1 protein sequence rather than by variations in the endoplasmic reticulum-folding environment. Moreover, we demonstrated direct effects of mutant protein trafficking on the response to small molecules that modulate the endoplasmic reticulum-folding environment by affecting Ca++ concentration. Finally, we developed a panel of isogenic human NPC1 iNeurons expressing WT, I1061T-, and R934L-NPC1 and demonstrated their utility in testing these candidate therapeutics. Our findings identify important rules governing mutant NPC1's response to proteostatic modulators and highlight the importance of species- and mutation-specific responses for therapy development.